The immune landscape of human thymic epithelial tumors

Human thymic epithelial tumors (TET) are common malignancies in the anterior mediastinum with limited biological understanding. Here we show, by single cell analysis of the immune landscape, that the developmental pattern of intra-tumoral T-cells identify three types within TETs. We characterize the developmental alterations and TCR repertoires of tumor-infiltrating T cells in the context of the distinguishing epithelial tumor cell types. We demonstrate that a subset of tumor cells, featuring medullary thymic epithelial cell (TEC) phenotype and marked by KRT14/GNB3 expression, accumulate in type 1 TETs, while T-cell positive selection is inhibited. Type 2 TETs are dominated by CCL25+ cortical TEC-like cells that appear to promote T-cell positive selection. Interestingly, the CHI3L1+ medullary TEC-like cells that are the characteristic feature of type 3 TETs don’t seem to support T-cell development, however, they may induce a tissue-resident CD8+ T cell response. In summary, our work suggests that the molecular subtype of epithelial tumour cells in TETs determine their tumour immune microenvironment, thus GNB3 and CHI3L1 might predict the immunological behavior and hence prognosis of these tumours.

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Reporting on sex and gender
The design of our study did not consider the effect of gender. The gender of the participants in our study was randomly determined. Our study did not focus on the effect of gender on TET tumors and therefore no sex-and gender-based analyses have been performed.

Recruitment
All patients with thymic epithelial tumors that were planed to undergo primary surgery could be asked to be recruited without selection. All patients provided written consent to participate in the study approval of local medical ethnics. There were no self-selection bias or other biases.

Ethics oversight
All samples were anonymously coded in accordance with local ethical guidelines (as stipulated by the Declaration of Helsinki), written informed consent was obtained, and the protocol was approved by the Review Board of the Second Affiliated Hospital of Zhejiang University School of Medicine.
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Sample size
In our study, a total of 42 patient samples were included. For CyTOF analysis, we included 22 patient samples and 3 normal samples. For scRNA-seq analysis, we included 6 patient samples and 1 normal samples. A total of 52,788 cells from tumors and 2,845 cells from the normal thymus were included for scRNA-seq analysis. For immuno-fluorescent staining, sections from 28 samples were included in the study.Flow cytometry was performed on 31 patient samples. In vitro co-culture experiment was performed on 6 patients.Sample size both for in vitro and in vivo was chosen taking in consideration the means of the target values between the experimental group and the control group, the standard error and the statistical analysis used. The sample size was able to ensure that our results were reliable.

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Data exclusions All cells expressing <500 genes were removed, as well as cells that contained <500 unique molecular identifiers (UMIs) and >25% mitochondrial counts.

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Experiments were replicated several times with reproducible results as indicated in figure legend/Statistics and reproducibility.
Randomization Samples were allocated to groups based on disease status (normal and tumor tissue) if applicable. The sample inclusion was random and there was no subjective allocation.

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In our study, blinding was not applicable to this study since it is exploratory in character and have no elements that might be influenced bybias from the subject or observer.
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